Nucleic acid ligands against infectious prions

ABSTRACT

The invention generally relates to nucleic acid ligands that specifically bind to infectious prions, and methods of diagnosing a transmissible spongiform encephalopathy disease in a subject. In certain embodiments, the invention provides an isolated nucleic acid ligand that binds to an infectious prion. In other embodiments, the invention provides a method for diagnosing a transmissible spongiform encephalopathy disease in a subject including obtaining a tissue or body fluid sample from a subject, contacting the tissue or body fluid with a nucleic acid ligand that binds to an infectious prion, thereby detecting the infectious prion in the sample, and diagnosing the transmissible spongiform encephalopathy disease based on results of the contacting step.

RELATED APPLICATION

This application is a divisional application and claims the benefit ofand priority to U.S. nonprovisional patent application Ser. No.12/611,473, filed Nov. 3, 2009, the entire content of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to nucleic acid ligands thatspecifically bind to infectious prions, and methods of diagnosing atransmissible spongiform encephalopathy disease in a subject.

BACKGROUND

Native or cellular prion protein, PrP^(C), is widely distributedthroughout mammals and has a particularly well-conserved amino acidsequence and protein structure. Infectious prions, PrP^(Sc), arecomposed of a modified form of the normal cellular prion protein. It isthought that a post-translational conformational change is involved inthe conversion of non-infectious PrP^(C) to infectious PrP^(Sc) duringwhich α-helices are transformed into β-sheets. PrP^(C) contains threeα-helices and has little β-sheet structure, while PrP^(Sc) is abundantin β-sheet. Infectious prions are resistant to proteases, the enzymes inthe body that can normally break down proteins.

The conversion of PrP^(C) to PrP^(Sc) leads to development oftransmissible spongiform encephalopathies (TSEs) during which PrP^(Sc)accumulates in the central nervous system and is accompanied byneuropathologic changes and neurological dysfunction. Infectious prionscause neurodegenerative disease by aggregating extracellularly withinthe central nervous system to form plaques known as amyloid that disruptthe normal tissue structure. This disruption is characterized by holesin the tissue with resultant spongy architecture due to vacuoleformation in neurons. Other histological changes include astrogliosisand absence of an inflammatory reaction. While the incubation period forprion diseases is generally quite long, once symptoms appear the diseaseprogresses rapidly, leading to brain damage and death. Neurodegenerativesymptoms can include convulsions, dementia, ataxia (balance andcoordination dysfunction), and behavioral or personality changes.

Specific examples of TSEs include scrapie, which affects sheep andgoats; bovine spongiform encephalopathy (BSE); transmissible minkencephalopathy, feline spongiform encephalopathy and chronic wastingdisease (CWD). In humans, TSE diseases may present themselves as kuru,Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker Syndrome(GSS), fatal insomnia, and variant Creutzfeldt-Jakob disease (vCJD).

No therapeutic agent exists to impede infectious prion propagation, andthus human TSE diseases are 100% fatal. A characteristic of all TSEs isthe lack of a measurable host immune response to the agent.Consequently, no antibodies specific for TSEs have been identified.Moreover, the lack of a known nucleic acid sequence precludes the use ofpolymerase chain reaction based diagnostic methods. Thus, noconventional serologic test may be used to identify infected animals.

Methodologies that can readily separate or that can distinguish betweentwo or more different conformational forms of a protein, e.g., PrP^(C)and PrP^(Sc), are needed to understand the process of conversion and tofind structures that will specifically interact with the diseaseassociated form. There also remains a need to identify high affinitybinding materials that are specific for the conformationally alteredprotein and especially forms associated with disease.

SUMMARY

The invention generally relates to nucleic acid ligands thatspecifically bind to infectious prions. A nucleic acid ligand (aptamer)is a nucleic acid macromolecule (e.g., DNA or RNA) that binds tightly toa specific molecular target. Like all nucleic acids, a particularnucleic acid ligand may be described by a linear sequence of nucleotides(A, U, T, C and G), typically 15-40 nucleotides long. Binding of anucleic acid ligand to a target molecule is not determined by nucleicacid base pairing. See, for example, Jayasena, Clin. Chem.45(9):1628-1650, 1999 and Baldrich et al., Anal. Chem. 2004;76(23):7053-7063. In solution, the chain of nucleotides formsintramolecular interactions that fold the molecule into a complexthree-dimensional shape. The shape of the nucleic acid ligand allows itto bind tightly against the surface of its target molecule. In additionto exhibiting remarkable specificity, nucleic acid ligands generallybind their targets with very high affinity, e.g., the majority ofanti-protein nucleic acid ligands have equilibrium dissociationconstants in the femtoomolar to low nanomolar range.

The nucleic acid ligands of the invention can distinguish betweennon-infection prions and infectious prions in a sample from patientsafflicted with human TSEs and animals afflicted with scrapie, BSE andCWD. Thus, nucleic acid ligands of the invention are useful fordetecting, binding to, isolating, removing, eliminating, extracting andseparating an infectious prion protein in or from a sample, such as abiological fluid or an environmental sample.

An aspect of the invention provides isolated nucleic acid ligands thatbind to an infectious prion. The nucleic acid ligands may be singlestranded or double stranded. The nucleic acid ligands may be DNA or RNA.In certain embodiments, the nucleic acid ligands include a nucleotidesequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2. In other embodiments, the nucleic acid ligands include an RNAsequence transcribed from a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2. The nucleic acid ligandsmay further include a detectable label, such as a fluorescent label.

Another aspect of the invention provides a method for diagnosing atransmissible spongiform encephalopathy disease (TSE) in a subjectincluding providing a tissue or body fluid sample from a subject,contacting the tissue or body fluid with a nucleic acid ligand thatbinds to an infectious prion, thereby detecting the infectious prion inthe sample, and diagnosing the transmissible spongiform encephalopathydisease based on results of the contacting step. The subject may be amammal such as a human.

TSEs that afflict humans include Creutzfeldt-Jakob Disease (CJD);Variant Creutzfeldt-Jakob Disease (vCJD); Gerstmann-Straussler-ScheinkerSyndrome; Fatal Familial Insomnia; and Kuru. TSEs that afflict othermammals, such as deer, pigs, cows, sheep, or goats, include BovineSpongiform Encephalopathy (BSE); Chronic Wasting Disease (CWD); Scrapie;Transmissible mink encephalopathy; Feline spongiform encephalopathy; andUngulate spongiform encephalopathy.

Another aspect of the invention provides isolated nucleotide sequencesincluding SEQ ID NO: 1 or SEQ ID NO: 2, or nucleotide sequencessubstantially identical thereto. The nucleotide sequences of theinvention may further include a detectable label, such as a fluorescentlabel. The isolated nucleotide sequences may bind to an infectiousprion.

Certain embodiments provide a polypeptide encoded by a nucleotidesequence including SEQ ID NO: 1, SEQ ID NO: 2, or nucleotide sequencessubstantially identical thereto. The invention also provides a vectorinclude a nucleotide sequence having SEQ ID NO: 1, SEQ ID NO: 2, ornucleotide sequences substantially identical thereto, operably linked toan expression control element. The invention also provides isolated hostcells transformed with a vector including a nucleotide sequence havingSEQ ID NO: 1, SEQ ID NO: 2, or nucleotide sequences substantiallyidentical thereto. Certain embodiments provide an isolated nucleotidesequence that hybridizes to a nucleotide sequence having SEQ ID NO: 1,SEQ ID NO: 2, or nucleotide sequences substantially identical theretounder high stringency conditions. Other embodiments provide an isolatednucleotide sequence that is complementary to a nucleotide sequencehaving SEQ ID NO: 1, SEQ ID NO: 2, or nucleotide sequences substantiallyidentical thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 panels A and B are photographs of a gel containing isolatednucleic acid ligands bound to infectious prions. Panel A shows a DNAtitration gel showing band density as function of DNA oligomerconcentration. Panel B shows a gel obtained by sequential applicationand electrophoresis of protein and DNA randomer solutions. The area ofPrP^(Sc) protein stained with EB due to presence of DNA is circled.

FIG. 2 is a photograph of a gel containing the extracted DNA nucleicacid ligands. Separation was obtained by PAGE electrophoresis.

FIG. 3 is a photograph of a gel showing PCR product. Lane A representsamplified starting DNA oligomers and Lane B represents amplified prionspecific oligomers.

FIG. 4 is a graph showing results of RT-PCR of nucleic acid ligands thatwere dissociated from PrP^(CWD).

FIG. 5 is a graph showing nucleic acid ligand detection of PrP^(CWD)using DNAse I treatment to remove unbound nucleic acid ligands.

FIG. 6 shows confirmation of specific PCR product after amplification ofthe nucleic acid ligands. Panel A is a graph showing a melt curveanalysis. Panel B shows an agarose gel electrophoresis.

DETAILED DESCRIPTION

The invention generally relates to nucleic acid ligands thatspecifically bind to infectious prions. This invention includes nucleicacid sequences that are substantially homologous to and that havesubstantially the same ability to bind infectious prions as the specificnucleic acid ligands shown in Examples below. Substantially homologousrefers to a degree of primary sequence homology in excess of about 70%,most preferably in excess of about 80%. Substantially the same abilityto bind an infectious prion refers to affinity within about two ordersof magnitude of the affinity of the ligands described herein. It is wellwithin the skill of those of ordinary skill in the art to determinewhether a given sequence is substantially homologous to thosespecifically described herein, and has substantially the same ability tobind an infectious prion.

The nucleic acid ligands may be single stranded or double stranded. Thenucleic acid ligands may be DNA or RNA. In certain embodiments, thenucleic acid ligands include a nucleotide sequence consisting of SEQ IDNO: 1 or SEQ ID NO: 2. In other embodiments, the nucleic acid ligandsinclude an RNA sequence transcribed from a nucleotide sequenceconsisting of SEQ ID NO: 1 or SEQ ID NO: 2.

Nucleic acid ligands of the invention were identified by methodsdescribed in co-pending and co-owned U.S. patent application Ser. No.12/611,436, filed Nov. 3, 2009, and entitled “Methods for identifyingnucleic acid ligands”, the contents of which are incorporated byreference herein in its entirety. Briefly, a candidate mixture ofnucleic acids is contacted to an infectious prion. After a sufficientincubation time, a single step selective separation protocol (e.g., gelelectrophoresis or HPLC gradient elution) is employed to eliminateundesirable competition for the infectious prion among nucleic acidsthat bind the infectious prion with greatest affinity, nucleic acidsthat bind the infectious prion with a lesser affinity, and nucleic acidsthat do not bind the infectious prion.

The selective separation protocols generate conditions in which thenucleic acids that bind the infectious prion with a lesser affinity andnucleic acids that do not bind the infectious prion cannot formcomplexes with the infectious prion or can only form complexes with theinfectious prion for a short period of time. In contrast, the conditionsof the separation protocols allow nucleic acids that bind the infectiousprion with greatest affinity to form complexes with the infectious prionand/or bind the infectious prion for the greatest period of time,thereby separating in a single step the nucleic acids with the greatestaffinity for the infectious prion, i.e., the nucleic acid ligands of theinvention, from the remainder of the nucleic acids of the candidatemixture.

An exemplary separation protocol includes loading the infectious prioninto a gradient gel, applying an electric current to cause theinfectious prion to migrate to a position in the gel, in which theinfectious prion remains immobilized at that position in the gel,loading the aptamer candidate mixture into the gel, and applying anelectric current to cause the candidate mixture to migrate through thegel, in which the nucleic acids with the greatest affinity for thetarget molecule (i.e., nucleic acid ligands of the invention) bind tothe infectious prion immobilized in the gel, and the nucleic acids withlesser affinity for the infectious prion and nucleic acids with noaffinity for the infectious prion migrate to an end of the gel. The gelsegment containing nucleic acid ligand/infectious prion complex is thencut from the gel, and the nucleic acid ligands are then dissociated fromthe infectious prion using a chaotropic agent.

Another exemplary separation protocol includes incubating the candidatemixture of nucleic acids with a plurality of infectious prions to formnucleic acid/infectious prion complexes, in which the infectious prionsare bound to beads, and eluting the nucleic acids from the complexesthat have been loaded onto an HPLC column by applying an HPLC gradientprofile, in which nucleic acids with the greatest affinity for theinfectious prion (i.e., nucleic acid ligands of the invention) elute atan end portion of the gradient profile and the nucleic acids with alesser affinity for the infectious prion and nucleic acids with noaffinity for the infectious prion elute prior to the end portion of thegradient profile. Many different HPLC gradient elution profiles areknown in the art. An exemplary HPLC gradient elution profile may includea linear increasing concentration of the infectious prion, in which anend portion of the gradient profile may include a linear increasingconcentration of the infectious prion and a chaotropic agent (e.g.,urea, guanidinium chloride, SCN⁻, or LiBr).

After separation, the nucleic acid ligands of the invention may besequenced. Sequencing may be by any method known in the art. See forexample Sanger et al. (Proc Natl Acad Sci USA, 74(12): 5463 67, 1977),Maxam et al. (Proc. Natl. Acad. Sci., 74: 560-564, 1977), and Drmanac,et al. (Nature Biotech., 16:54-58, 1998), which references describeexemplary conventional ensemble sequencing techniques. Also see Lapiduset al. (U.S. Pat. No. 7,169,560), Quake et al. (U.S. Pat. No.6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S. patentapplication number 2002/0164629), and Braslaysky, et al., (PNAS (USA),100: 3960-3964, 2003), which references describe exemplary singlemolecule sequencing by synthesis techniques. The contents of each of thereferences is incorporated by reference herein in its entirety.

The nucleic acid ligands may further include a detectable label, such asradioactive labels, chemoluminescent labels, luminescent labels,phosphorescent labels, fluorescence polarization labels, and chargelabels.

In certain embodiments, the detectable label is a fluorescent label.Suitable fluorescent labels include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

The fluorescently labeled nucleotides may be obtained commercially(e.g., from NEN DuPont, Amersham, and BDL). Alternatively, fluorescentlylabeled nucleotides may also be produced by various techniques, such asthose described in Kambara et al. (Bio/Technol., 6:816-21, 1988); Smithet al. (Nucl. Acid Res., 13:2399-2412, 1985); and Smith et al. (Nature,321: 674-679, 1986). The fluorescent dye may be linked to thedeoxyribose by a linker arm that is easily cleaved by chemical orenzymatic means. There are numerous linkers and methods for attachinglabels to nucleotides, as shown in Oligonucleotides and Analogues: APractical Approach (IRL Press, Oxford, 1991); Zuckerman et al.(Polynucleotides Res., 15: 5305-5321, 1987); Sharma et al.(Polynucleotides Res., 19:3019, 1991); Giusti et al. (PCR Methods andApplications, 2:223-227, 1993); Fung et al. (U.S. Pat. No. 4,757,141);Stabinsky (U.S. Pat. No. 4,739,044); Agrawal et al. (TetrahedronLetters, 31:1543-1546, 1990); Sproat et al. (Polynucleotides Res.,15:4837, 1987); and Nelson et al. (Polynucleotides Res., 17:7187-7194,1989). Extensive guidance exists in the literature for derivatizingfluorophore and quencher molecules for covalent attachment via commonreactive groups that may be added to a nucleotide. Many linking moietiesand methods for attaching fluorophore moieties to nucleotides alsoexist, as described in Oligonucleotides and Analogues, supra; Guisti etal., supra; Agrawal et al, supra; and Sproat et al., supra.

Certain chemical modifications of the nucleic acid ligands of theinvention may be made to increase the in vivo stability of the nucleicacid ligand or to enhance or to mediate the delivery of the nucleic acidligand. See, e.g., Pieken et al. (U.S. Pat. No. 5,660,985), the contentsof which are incorporated by reference herein in their entirety.Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those that provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to2-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping. In certainembodiments of the instant invention, the nucleic acid ligands are RNAmolecules that are 2′-fluoro (2′-F) modified on the sugar moiety ofpyrimidine residues.

Nucleic acid ligands of the invention can further include a nucleotideanalog. Exemplary nucleotide analogs include xanthine or hypoxanthine,5-bromouracil, 2-aminopurine, deoxyinosine, or methylated cytosine, suchas 5-methylcytosine, N4-methoxydeoxycytosine, and the like. Alsoincluded are bases of polynucleotide mimetics, such as methylatednucleic acids, e.g., 2′-O-methRNA, peptide nucleic acids, locked nucleicacids, modified peptide nucleic acids, and any other structural moietythat acts substantially like a nucleotide or base, for example, byexhibiting base-complementarity with one or more bases that occur in DNAor RNA.

The nucleic acid ligands of the invention are capable of distinguishingbetween different conformational forms of PrP^(C) and PrP^(Sc), and thusprovide high affinity binding materials that are specific for theconformationally altered protein and especially forms associated withdisease. Accordingly, another aspect of the invention provides methodsfor diagnosing any disease in a subject associated with an infectiousprion. In certain embodiments, the invention provides a method fordiagnosing a transmissible spongiform encephalopathy disease (TSE) in asubject. The subject may be a mammal such as a human. TSEs that afflicthumans include Creutzfeldt-Jakob Disease (CJD); VariantCreutzfeldt-Jakob Disease (vCJD); Gerstmann-Straussler-ScheinkerSyndrome; Fatal Familial Insomnia; and Kuru. TSEs that afflict othermammals, such as deer, pigs, cows, sheep, or goats, include BovineSpongiform Encephalopathy (BSE); Chronic Wasting Disease (CWD); Scrapie;Transmissible mink encephalopathy; Feline spongiform encephalopathy; andUngulate spongiform encephalopathy.

Methods of the invention include providing a tissue or body fluid samplefrom a subject. A sample may originate from a mammal, e.g. a human, andincludes tissue or body fluid. The sample may also originate from othermammals, such as deer, pigs, cows, sheep, or goats. A tissue is a massof connected cells and/or extracellular matrix material, e.g. skintissue, nasal passage tissue, CNS tissue, neural tissue, eye tissue,liver tissue, kidney tissue, placental tissue, mammary gland tissue,gastrointestinal tissue, musculoskeletal tissue, genitourinary tissue,bone marrow, and the like, derived from, for example, a human or othermammal and includes the connecting material and the liquid material inassociation with the cells and/or tissues. A body fluid is a liquidmaterial derived from, for example, a human or other mammal. Such bodyfluids include, but are not limited to urine, sputum, stool, mucous,saliva, blood, plasma, serum, serum derivatives, bile, phlegm, sweat,amniotic fluid, mammary fluid, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A sample may also be a fine needle aspirateor biopsied tissue. A sample also may be media containing cells orbiological material.

Methods of the invention further include contacting the tissue or bodyfluid with a nucleic acid ligand that binds to an infectious prion,thereby detecting the infectious prion in the sample. Nucleic acidligand-based assays are very similar to antibody-based assays but use anucleic acid ligand instead of an antibody. Similar to an antibody, anucleic acid ligand has a molecular activity such as having an enzymaticactivity or binding to a target molecule at a specific region (i.e.,similar to an epitope for an antibody). Thus, any suitableantibody-based technique may be adapted for use with the presentmethods. Exemplary antibody-based assays that can be adapted for usewith nucleic acid ligands include Western blot analysis,radioimmunoassay (RIA), immunofluorescent assay, chemiluminescent assay,or enzyme-linked immunosorbent assay (ELISA). Protocols of these assaysare described in Stenman et al. (U.S. Pat. No. 5,976,809), Tamarkin etal. (U.S. Pat. No. 5,965,379), Chen (U.S. Pat. No. 5,571,680), Griffinet al. (U.S. Pat. No. 5,279,956), and Price et al. (U.S. Pat. No.6,579,684), the contents of each of which are incorporated by referenceherein in its entirety. Adaptation of these antibody-based assays foruse with nucleic acid ligands is well within the knowledge of one ofskill in the art.

An exemplary antibody-based assay that has been adapted for use with anucleic acid ligand is an ALISA (Aptamer-Linked Immobilized SorbentAssay) procedure, described in Vivekananda et al. (Lab Invest.86(6):610-618, 2006), the contents of which are incorporated byreference herein in its entirety. Based on results of the assay, thetransmissible spongiform encephalopathy disease is diagnosed.

Another aspect of the invention provides isolated nucleotide sequencesincluding SEQ ID NO: 1 or SEQ ID NO: 2, or nucleotide sequencessubstantially identical thereto. Examples below describe isolation andpurification of the nucleotide sequences of the invention. A nucleicacid or fragment thereof has substantial identity with another if, whenoptimally aligned (with appropriate nucleotide insertions or deletions)with the other nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 60% of the nucleotidebases, usually at least about 70%, more usually at least about 80%,preferably at least about 90%, and more preferably at least about 95-98%of the nucleotide bases.

Identity refers to degree of sequence relatedness between twopolynucleotide sequences as determined by the identity of the matchbetween two strings of such sequences, such as the full and completesequence. Identity can be readily calculated. While there exist a numberof methods to measure identity between two polynucleotide sequences, theterm identity is well known to skilled artisans (see e.g., ComputationalMolecular Biolog, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991).

Methods commonly employed to determine identity between two sequencesinclude, but are not limited to, those shown in Guide to Huge Computers,Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H.,and Lipman, D., SIAM J Applied Math. 48:1073 (1988). In certainembodiments, methods to determine identity are designed to give thelargest match between the two sequences tested. Such methods arecodified in computer programs. Exemplary computer program methods todetermine identity between two sequences include, but are not limitedto, GCG (Genetics Computer Group, Madison Wis.) program package(Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP,BLASTN, FASTA (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410;and Altschul, S. F. et al. (1997) Nucl. Acids Res. 25:3389-3402). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Aspects of the invention also include nucleotide sequences thathybridize to SEQ ID NO: 1 or SEQ ID NO: 2 under high stringencyconditions. Nucleic acid hybridization may be affected by suchconditions as salt concentration, temperature, or organic solvents, inaddition to base composition, length of complementary strands, andnumber of nucleotide base mismatches between hybridizing nucleic acids,as is readily appreciated by those skilled in the art. Stringency ofhybridization reactions is readily determinable by one of ordinary skillin the art, and generally is an empirical calculation dependent uponsequence length, washing temperature, and salt concentration. Ingeneral, longer sequences require higher temperatures for properannealing, while shorter sequences need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowits melting temperature. The higher the degree of desired homologybetween the sequence and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995), the contents of which are incorporated by referenceherein in their entirety.

Stringent conditions or high stringency conditions typically: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1%SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

Moderately stringent conditions may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989 (the contents of which are incorporated byreference herein in their entirety), and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent that those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37° C. to 50° C. Theskilled artisan will recognize how to adjust the temperature, ionicstrength, etc. as necessary to accommodate factors such as sequencelength and the like.

Isolated nucleotide sequences of the invention may be inserted intovectors for expression in an isolated host cell. In accordance with thepresent invention nucleotide sequences of the invention may beengineered into recombinant DNA molecules to direct expression of apolypeptide encoded by SEQ ID NO: 1 or SEQ ID NO: 2 in appropriate hostcells. To express a biologically active polypeptide, a nucleotidesequence encoding the polypeptide, e.g., SEQ ID NO: 1 or SEQ ID NO: 2,or functional equivalent, is inserted into an appropriate expressionvector, i.e., a vector that contains the necessary nucleic acid encodingelements that regulate transcription and translation of the insertedcoding sequence, operably linked to the nucleotide sequence encoding thepolypeptide amino acid sequence.

Methods that are well known to those skilled in the art are used toconstruct expression vectors containing a sequence encoding thepolypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 operably linked toappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques and in vivo recombination or genetic recombination. Suchtechniques are described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989.

A variety of commercially available expression vector/host systems areuseful to contain and express a polypeptide encoded by SEQ ID NO: 1 orSEQ ID NO: 2. These include but are not limited to microorganisms suchas bacteria transformed with recombinant bacteriophage, plasmid orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems contacted with virus expression vectors(e.g., baculovirus); plant cell systems transfected with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with bacterial expression vectors (e.g., Ti,pBR322, or pET25b plasmid); or animal cell systems. See Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,N.Y., 1989.

Virus vectors include, but are not limited to, adenovirus vectors,lentivirus vectors, adeno-associated virus (AAV) vectors, andhelper-dependent adenovirus vectors. Adenovirus packaging vectors arecommercially available from American Type Tissue Culture Collection(Manassas, Va.). Methods of constructing adenovirus vectors and usingadenovirus vectors are shown in Klein et al., Ophthalmology,114:253-262, 2007 and van Leeuwen et al., Eur. J. Epidemiol.,18:845-854, 2003. Adenovirus vectors have been used in eukaryotic geneexpression (Levrero et al., Gene, 101:195-202, 1991) and vaccinedevelopment (Graham et al., Methods in Molecular Biology: Gene Transferand Expression Protocols 7, (Murray, Ed.), Humana Press, Clifton, N.J.,109-128, 1991). Further, recombinant adenovirus vectors are used forgene therapy (Wu et al., U.S. Pat. No. 7,235,391).

Recombinant adenovirus vectors are generated, for example, fromhomologous recombination between a shuttle vector and a provirus vector(Wu et al., U.S. Pat. No. 7,235,391). The adenovirus vectors herein arereplication defective, for example, are conditionally defective, lackingadenovirus E1 region, and a polynucleotide of SEQ ID NO: 1 or SEQ ID NO:2 is introduced at the position from which the E1-coding sequences havebeen removed. The polynucleotide sequence alternatively is inserted inthe E3 region, or is inserted in an E4 region using a helper cell line.

Helper cell lines may be derived from human cells such as, 293 humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus, e.g., Vero cells or other monkeyembryonic mesenchymal or epithelial cells. Generation and propagation ofthese replication defective adenovirus vectors using a helper cell lineis described in Graham et al, J. Gen. Virol., 36:59-72, 1977.

Lentiviral vector packaging vectors are commercially available fromInvitrogen Corporation (Carlsbad Calif.). An HIV-based packaging systemfor the production of lentiviral vectors is prepared using constructs inNaldini et al., Science 272: 263-267, 1996; Zufferey et al., NatureBiotechnol., 15: 871-875, 1997; and Dull et al., J. Virol. 72:8463-8471, 1998.

A number of vector constructs are available to be packaged using asystem, based on third-generation lentiviral SIN vector backbone (Dullet al., J. Virol. 72: 8463-8471, 1998). For example the vector constructpRRLsinCMVGFPpre contains a 5′ LTR in which the HIV promoter sequencehas been replaced with that of Rous sarcoma virus (RSV), aself-inactivating 3′ LTR containing a deletion in the U3 promoterregion, the HIV packaging signal, RRE sequences linked to a marker genecassette consisting of the Aequora jellyfish green fluorescent protein(GFP) driven by the CMV promoter, and the woodchuck hepatitis virus PREelement, which appears to enhance nuclear export. The GFP marker geneallows quantitation of transfection or transduction efficiency by directobservation of UV fluorescence microscopy or flow cytometry (Kafri etal., Nature Genet., 17: 314-317, 1997 and Sakoda et al., J. Mol. Cell.Cardiol., 31: 2037-2047, 1999).

Manipulation of retroviral nucleic acids to construct a retroviralvector containing the SEQ ID NO: 1 or SEQ ID NO: 2 and packaging cellsis accomplished using techniques known in the art. See Ausubel, et al.,1992, Volume 1, Section III (units 9.10.1-9.14.3); Sambrook, et al.,1989. Molecular Cloning: A Laboratory Manual. Second Edition. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Miller, etal., Biotechniques. 7:981-990, 1989; Eglitis, et al., Biotechniques.6:608-614, 1988; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289,5,122,767, and 5,124,263; and PCT patent publications numbers WO85/05629, WO 89/07150, WO 90/02797, WO 90/02806, WO 90/13641, WO92/05266, WO 92/07943, WO 92/14829, and WO 93/14188.

A retroviral vector is constructed and packaged into non-infectioustransducing viral particles (virions) using an amphotropic packagingsystem. Examples of such packaging systems are found in, for example,Miller, et al., Mol. Cell. Biol. 6:2895-2902, 1986; Markowitz, et al.,J. Virol. 62:1120-1124, 1988; Cosset, et al., J. Virol. 64:1070-1078,1990; U.S. Pat. Nos. 4,650,764, 4,861,719, 4,980,289, 5,122,767, and5,124,263, and PCT patent publications numbers WO 85/05629, WO 89/07150,WO 90/02797, WO 90/02806, WO 90/13641, WO 92/05266, WO 92/07943, WO92/14829, and WO 93/14188.

Generation of “producer cells” is accomplished by introducing retroviralvectors into the packaging cells. Examples of such retroviral vectorsare found in, for example, Korman, et al., Proc. Natl. Acad. Sci. USA.84:2150-2154, 1987; Morgenstern, et al., Nucleic Acids Res.18:3587-3596, 1990; U.S. Pat. Nos. 4,405,712, 4,980,289, and 5,112,767;and PCT patent publications numbers WO 85/05629, WO 90/02797, and WO92/07943.

Herpesvirus packaging vectors are commercially available from InvitrogenCorporation, (Carlsbad, Calif.). Exemplary herpesviruses are anα-herpesvirus, such as Varicella-Zoster virus or pseudorabies virus; aherpes simplex virus such as HSV-1 or HSV-2; or a herpesvirus such asEpstein-Ban virus. A method for preparing empty herpesvirus particlesthat can be packaged with a desired nucleotide segment, for example anucleotide seuqnece include SEQ ID NO: 1 or SEQ ID NO: 2, in the absenceof a helper virus that is capable to most herpesviruses is shown inFraefel et al. (U.S. Pat. No. 5,998,208, issued Dec. 7, 1999).

The herpesvirus DNA vector can be constructed using techniques familiarto the skilled artisan. For example, DNA segments encoding the entiregenome of a herpesvirus is divided among a number of vectors capable ofcarrying large DNA segments, e.g., cosmids (Evans, et al., Gene 79,9-20, 1989), yeast artificial chromosomes (YACS) (Sambrook, J. et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989) or E. coli F element plasmids(O'Conner, et al., Science 244:1307-1313, 1989).

For example, sets of cosmids have been isolated which containoverlapping clones that represent the entire genomes of a variety ofherpesviruses including Epstein-Barr virus, Varicella-Zoster virus,pseudorabies virus and HSV-1. See M. van Zijl et al., J. Virol. 62,2191, 1988; Cohen, et al., Proc. Nat'l Acad. Sci. U.S.A. 90, 7376, 1993;Tomkinson, et al., J. Virol. 67, 7298, 1993; and Cunningham et al.,Virology 197, 116, 1993.

AAV is a dependent parvovirus in that it depends on co-infection withanother virus (either adenovirus or a member of the herpes virus family)to undergo a productive infection in cultured cells (Muzyczka, Curr TopMicrobiol Immunol, 158:97 129, 1992). For example, recombinant AAV(rAAV) virus is made by co-transfecting a plasmid containing the gene ofinterest, for example, the CD59 gene, flanked by the two AAV terminalrepeats (McLaughlin et al., J. Virol., 62(6):1963 1973, 1988; Samulskiet al., J. Virol, 63:3822 3828, 1989) and an expression plasmidcontaining the wild-type AAV coding sequences without the terminalrepeats. Cells are also contacted or transfected with adenovirus orplasmids carrying the adenovirus genes required for AAV helper function.Recombinant AAV virus stocks made in such fashion include withadenovirus which must be physically separated from the recombinant AAVparticles (for example, by cesium chloride density centrifugation).

Adeno-associated virus (AAV) packaging vectors are commerciallyavailable from GeneDetect (Auckland, New Zealand). AAV has been shown tohave a high frequency of integration and infects nondividing cells, thusmaking it useful for delivery of genes into mammalian cells in tissueculture (Muzyczka, Curr Top Microbiol Immunol, 158:97 129, 1992). AAVhas a broad host range for infectivity (Tratschin et al., Mol. Cell.Biol., 4:2072 2081, 1984; Laughlin et al., J. Virol., 60(2):515 524,1986; Lebkowski et al., Mol. Cell. Biol., 8(10):3988 3996, 1988;McLaughlin et al., J. Virol., 62(6):1963 1973, 1988).

Methods of constructing AAV vectors and using AAV vectors are described,for example in U.S. Pat. Nos. 5,139,941 and 4,797,368. Use of AAV ingene delivery is further described in LaFace et al., Virology,162(2):483 486, 1988; Zhou et al., Exp. Hematol, 21:928 933, 1993;Flotte et al., Am. J. Respir. Cell Mol. Biol., 7(3):349 356, 1992; andWalsh et al., J. Clin. Invest, 94:1440 1448, 1994.

Recombinant AAV vectors have been used successfully for in vitro and invivo transduction of marker genes (Kaplitt et al., Nat. Genet., 8(2):14854, 1994; Lebkowski et al., Mol. Cell. Biol., 8(10):3988 3996, 1988;Samulski et al., EMBO J., 10:3941 3950, 1991; Shelling and Smith, GeneTherapy, 1: 165 169, 1994; Yoder et al., Blood, 82 (Supp.): 1:347A,1994; Zhou et al., Exp. Hematol, 21:928 933, 1993; Tratschin et al.,Mol. Cell. Biol., 5:3258 3260, 1985; McLaughlin et al., J. Virol.,62(6):1963 1973, 1988) and transduction of genes involved in humandiseases (Flotte et al., Am. J. Respir. Cell Mol. Biol., 7(3):349 356,1992; Ohi et al., Gene, 89(2):279 282, 1990; Walsh et al., J. Clin.Invest, 94:1440 1448, 1994; and Wei et al., Gene Therapy, 1:261268,1994).

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Identifying Nucleic Acid Ligands Using GelElectrophoresis

Preparation of Candidate Mixture

DNA oligomers were obtained from a well established library of PCRamplifiable DNA randomers described in Vivekananda et al. (Lab Invest.86(6):610-618, 2006). This library was custom manufactured atSigma-Genosys (The Woodlands, Tex.).

Nucleic Acid Ligand Capture

A solution containing infectious prion protein that resulted in chronicwaste disease (PrP^(CWD)) was loaded into each well of a gradient gel.Native PAGE electrophoresis was performed to immobilize the infectiousprion at a position in the gel. After immobilization of the infectiousprion at a position in the gel, the concentrated solution of DNArandomers was loaded into each well of the gel. Electrophoresis wasperformed for a second time so that the DNA randomers would migratethrough the gel already having the infectious prions immobilized at aposition in the gel. Per each well, 25 μL of the prion protein solutionand 25 μL of DNA oligomer solution containing 3.9 μg DNA weresequentially applied and electrophoretically resolved.

During the second electrophoretic step, the PrP^(CWD) proteins barelymoved in the electrophoretic field and stayed trapped in the network ofthe gradient gel, while the small DNA oligomers (MW of 21 kDa) were ableto migrate towards the positive electrode. Only those DNA oligomers thatshowed high affinity to PrP^(CWD) and could withstand the effect ofdilution by the running buffer and the effect of the electrostatic fieldremained attached to the target protein. Remaining DNA randomersmigrated past the PrP^(CWD) to an end of the gel.

After the second electrophoretic step, the gels were stained withEthidium Bromide (EB) to detect and quantify DNA oligomer bound to thePrP^(CWD) protein within the gel. After performing the above procedureretention of some small quantities of DNA oligonucleotides located onthe PrP^(CWD) entrapped in the gel was observed (FIG. 1). Panel A ofFIG. 1 is a control showing an electrophoresis run of only DNArandomers, no protein. Panel B shows gels obtained by sequentialapplication and electrophoresis of protein and DNA randomer solutions.The area of DNA randomer/PrP^(CWD) complexes stained with EB due topresence of DNA is circled.

As shown in FIG. 1 panel B, sub ng quantities of DNA were observed inthe area of localization of DNA randomers complexed with PrP^(CWD)protein. This allowed collection and amplification/sequencing of thisDNA fraction as a potential candidate for PrP^(CWD) detection. Theestimate of the affinity of the retained aptamer was provided using theestimated amounts of PrP^(CWD) (˜400 ng, FIG. 1) and nucleic acid ligand(˜0.18 ng, FIG. 2), and average molecular weight of the PrP^(CWD)protein of 275×10³ g/mol. Thus, the dissociation constant was determinedby Equation 1.

K _(d)=[PrP^(CWD)]×[Apt]/[PrP^(CWD)Apt]/MWPr^(CWD)protein,  Equation 1

where: [PrP^(CWD)] is the equilibrium quantity of the prion protein(ng); [Apt] is the equilibrium quantity of the retained aptamer (ng);[PrP^(CWD)Apt] is the is the equilibrium quantity of the complex of theprion protein and retained aptamer (ng); and MWPrP^(CWD)protein is themolecular weight of the detected MWPrP^(CWD)protein ng/nmol.

K _(d)=400×0.18/418/(275×10³);

K _(d)=6×10⁻⁵ nmol

Nucleic Acid Ligand Isolation

In order to obtain protein free aptamers via PAGE, the zones containingnucleic acid ligands bound to PrP^(CWD) were carefully cut from thepolyacrylamide gels and treated with chaotropic agent (2.0 M solution ofsodium thiocyanate, NaSCN). A total of three 1×8 cm gel fragments wereimmersed in 10 mL of the NaSCN solution in a 50 mL Falcon tube andvortexed for 1 hr at ˜1000 rpm. The resultant gel-water suspension wasfiltered through a 0.8 micron cellulose acetate syringe filter. Thefiltrate was analyzed for the presence of DNA aptamers by PAGE (4-20%gradient gel) with silver staining according to the protocol provided byBioRad Laboratories. DNA 70-randomer was used to serve as standards forthis run. As seen in FIG. 2, the DNA nucleic acid ligands were dilutedgiven the faint bands appearing in the gel.

Nucleic Acid Ligand Amplification

The nucleic acid ligands were amplified by PCR in a G-storm thermalcycler at IST. More specifically, PCR amplification was performed byadding the collected nucleic acid ligands, primers, and nuclease freewater to the EconoTaq 2× Master Mix (Table 1) and followingamplification steps depicted in Table 2.

TABLE 1 PCR reagent composition Amount Final Reagent (μl) ConcentrationConcentration Lucigen Econotaq 25 2X 1X 2X Master Mix Primer AP7: 1.0 50pmol/μl 1 pmol/μl TAC GAC TCA CTA TAG GGA TCC (SEQ ID NO: 3) Primer AP3:1.0 50 pmol/μl 1 pmol/μl AAC CCT CAC TAA AGG GAA TT (SEQ ID NO: 4)Nucleic acid ligand: 1.0 ~1 ng/μl <1 ng/μl 5′-TAC GAC TCA CTA TAG GGATCC- (N = 28) -GAA TTC CCT TTA GTG AGG GTT-3′ (SEQ ID NO: 5) NucleaseFree Water 22.0 — —

TABLE 2 PCR steps Cycling step Temperature (° C.) Time # of CyclesInitial Denaturation 95 2 min 1 Denaturation 95 30 sec 30 Annealing*50-65 *52.9 30 sec 30 Extension 72 30 sec 30 Final Extension 72 5 min 1Hold  4 overnight 1 *Optimum annealing temperatureIn this manner 4.0 mL solution containing 100 μg of >80% pure nucleicacid ligands was generated (FIG. 3, Lane B).

Nucleic Acid Ligand Identification

The PCR amplification provided amplified material that was sequenced atSequegen Inc, Worcester, Mass. The obtained nucleic acid ligandsequences were used to generate micromole quantities of the purifiednucleic acid ligand sequences at Sigma-Genosys (The Woodlands, Tex.).The sequences of the obtained nucleic acid ligands were as follows:

(SEQ ID NO: 1) TACGACTCACTATAGGGATCCGTTTTTCCGTACTTCTTAAATCGAATTCCCTTTAGTGAGGGTT;  (SEQ ID NO: 2)TACGACTCACTATAGGGATCCTTCTCCGCACTACTTTACCTCGAGTGCT ATTCCCTTTAGTGAGGGTT.

Nucleic Acid Ligand Affinity and Specificity Assay

To evaluate the specificity of the generated DNA nucleic acid ligands,quantitative real-time PCR (qrt-PCR) was used to quantify low amounts ofbound nucleic acid ligand from PrP^(CWD) positive biological samples.The following process was used:

-   -   1. Diluted the elk strain of CWD prions (designated as E2) used        in the nucleic acid ligand isolation protocol in PBS from 1:10        to 1:100,000.    -   2. 1:10 dilutions of uninfected normal brain homogenate (NBH)        were used as negative controls.    -   3. 20 μL of each dilution sample was incubated with 180 μL of 1        μM of two different nucleic acid ligand (SEQ ID NO: 1 and SEQ ID        NO: 2) for twenty minutes at room temperature.    -   4. Unbound nucleic acid ligands were removed from the samples by        dilution with 300 μL PBS and filtration through a 100 Kd spin        column.    -   5. Step 4 was repeated twice, substituting 500 μL 500 mM NaSCN        for PBS.    -   6. Samples were concentrated to fifty microliters, and nucleic        acid ligands purified using the DNeasy blood and tissue kit        (Qiagen).    -   7. Nucleic acid ligands were eluted from columns with 50 μL        Tris-EDTA buffer and ten microliters used for nucleic acid        ligand detection by real-time PCR using primers specific for        nucleic acid ligands and SYBR Green fluorescent        DNA-intercalating dye.    -   8. Two HOH-only samples were used as negative controls for PCR.    -   9. All samples were incubated for one pre-melt cycle for three        minutes at 95° C., then thirty cycles of twenty seconds at        95° C. and 45 seconds at 50° C. for annealing and extension and        a final cycle for five minutes at 50° C.

After forty PCR cycles, both negative HOH controls (cyan and greentraces (FIG. 4) and Table 3) remained below fluorescent detectionthreshold (orange horizontal line). The 1:10 E2 dilutions were off thescale where the fluorescence signal was detected without the need foramplification (C_(T)=0). All other E2 dilutions were detected with C_(T)values of 11.7 for 1:100 to 20.3 for 1:100 000 dilutions. Backgrounddetection of nucleic acid ligands eluting with the NBH dilutions (redand blue traces, FIG. 4) was easily distinguished from all othersamples, which were well above 1000 relative fluorescence units. Table 3summarizes the results shown in FIG. 4.

TABLE 3 C_(T) values for RT-PCR samples Well Identifier Ct A01 HOH N/AA02 HOH N/A B01 1:10 E2 (SEQ ID NO: 1) 0 B02 1:10 E2 (SEQ ID NO: 2) 0C01 1:100 E2 (SEQ ID NO: 1) 11.7 C02 1:100 E2 (SEQ ID NO: 2) 11.4 D011:1000 E2 (SEQ ID NO: 1) 11.4 D02 1:1000 E2 (SEQ ID NO: 2) 12.3 E011:10,000 E2 (SEQ ID NO: 1) 13.3 E02 1:10,000 E2 (SEQ ID NO: 2) 15.7 F011:100,000 E2 (SEQ ID NO: 1) 20.3 F02 1:100,000 E2 (SEQ ID NO: 2) 16.4G01 1:10 NBH (SEQ ID NO: 1) 17.4 G02 1:10 NBH (SEQ ID NO: 2) 28.4

In Table 3, the C_(T) values represented the number of cycles requiredfor the fluorescence signal to cross the set threshold. A C_(T) value≦29 indicated an abundance of the target, a C_(T) value of 30-37indicated a positive detection of the target, and a C_(T) value of 38-40indicated the presence of a weak or small amount of the target.

This data show that the two nucleic acid ligands identified by methodsof the invention successfully detected up to a 100,000-fold dilution ofE2 CWD prions. This detection threshold is at least 100-fold greaterthan detection by conventional western blotting. Further advances inoptimization of the assay to achieve a detection limit to 1,000,000dilutions of the prion sample are described below.

To simulate the real low pg and fg concentrations of infectious prionsin bodily fluids, the affinity of the DNA nucleic acid ligands wasfurther evaluated at 1:100,000 dilutions of brain homogenate (actualworking dilution of 1.25×10⁶ under the established PCR protocol). Thisdilution level represents a target sample concentration 0.3 pg/mL ofPrP^(Sc) in the tested brain homogenate which is well within the rangeof infectious prion levels found in the blood of live animals (Brown etal., J Lab Clin Med, 137(1):5-13, 2001). Therefore, this sensitivityprovides for tests to be conducted on bio-fluids collected from liveanimals. For example, the concentration of the infectious prions in theblood of infected animals can be estimated at sub pg/mL levels, whilethe threshold of sensitivity for the detection of infectious prionsextracted from scrapie-infected hamster brain has been estimated at 5pg/mL (Brown et al., J Lab Clin Med, 137(1):5-13, 2001). The estimatedconcentration of PrP^(Sc) in the urine of infected test animals has beenestimated in the fg/mL (0.001 pg) levels (Gonzalez-Romero et al., FEBSLett, 582(21-22):3161-3166, 2008). Therefore, the DNA nucleic acidligands developed above can be used to detect infectious prions in about10 μL of blood or 1 mL of urine.

Data obtained at the 1,000,000 dilutions showed that the infectiousprions were detected (total 1.25×10⁶ dilution) of the tested specimen.This corresponds to significant improvement of the detection limit to0.025 pg/mL or 250 atto-grams in a 10 μL sample.

The detection of infectious prions in the 1:100,000 dilutions (C_(T) of16.4-20.3) are comparable to the low-level detection of nucleic acidligands in the NBH samples (blue and red traces, C_(T) of 17.4-28.4)which were at 1:10 dilutions. In addition, the relative fluorescentsignals of the NBH samples were much lower and easily distinguishablefrom test samples (see FIG. 4). These data also showed that optimizationremoves the low-level detection of the NBH sample altogether. Thefluorescence signal of the NBH samples at 1:100 dilutions would beextremely weak to non-existent.

To completely eliminate background nucleic acid ligand noise in thesamples, the samples were further treated with ten units of DNAse I forone minute to remove unbound nucleic acid ligands, while PrP^(CWD)-boundnucleic acid ligands were protected from digestion. Samples were thentreated with 50 μg/mL proteinase K (PK) to eliminate DNAse I activity,then heat inactivated PK for ten minutes at 95° C. to maintain integrityand activity of the Taq polymerase used in the RT-PCR amplification ofbound nucleic acid ligands. This DNAse I/PK/heat inactivation protocolcompletely eliminated possible background amplification of nucleic acidligands in our negative control samples (FIG. 5) and obviated the neednot only for NaSCN treatment and filtration, but also for nucleic acidligand purification using the DNEasy tissue kit. The DNAse I and PKtreatment greatly decreased the organic complexity of the samples, whichcould be used directly in the QRT-PCR reaction without the time andexpense of further DNA extraction. The entire assay was completed fromnucleic acid ligand incubation to RT-PCR to data analysis, in less thanthree hours. CWD prions were successfully detected in a 10⁻⁶ dilution ofbrain homogenate from a CWD-infected elk, corresponding to athousand-fold increase in sensitivity over conventional proteinase Kdigestion/western blotting. Data also show prion detection in archivedspleen tissue from mice infected with CWD (Table 4).

TABLE 4 Summary of additional data generated from subsequent bindingassays Sample Prion¹ Dilution² Detection³ Brain Spiked 10⁻² + BrainSpiked 10⁻³ + Brain Spiked 10⁻⁴ + Brain Spiked 10⁻⁵ + Brain Spiked10⁻⁶ + Brain Infected 10⁻³ + Brain Negative 10⁻¹ − Brain Negative 10⁻² −Brain Negative 10⁻³ − Spleen Infected 10⁻¹ + Spleen Negative 10⁻¹ −

Generation of the specific nucleic acid ligand PCR products wereconfirmed by both melt curve analysis (FIG. 6 panel A) and agarose gelelectrophoresis (FIG. 6 panel B) of amplified samples.

Data herein show that the selected DNA nucleic acid ligands generated bymethods of the invention could detect infectious prions at levels as lowas 0.03 pg/mL concentrations directly in 20 μL samples of biologicalspecimens. This sensitivity is at least 1000-fold higher than what isachievable in immuno-enzymatic assays. Data herein further show thespecificity of these DNA nucleic acid ligands for infectious prions overnormal prions. There were no false positive or negative reactions incontrols containing normal prion protein or no prion protein.

These results show that DNA nucleic acid ligands generated by methods ofthe invention can detect very low concentrations of infectious prionthat are representative of concentrations found in biological fluids orsamples such as blood, urine and feces.

1-8. (canceled)
 9. A method for diagnosing a transmissible spongiformencephalopathy disease in a subject comprising: providing a tissue orbody fluid sample from a subject; contacting the tissue or body fluidwith a nucleic acid ligand that binds to an infectious prion, therebydetecting the infectious prion in the sample; and diagnosing thetransmissible spongiform encephalopathy disease based on results of thecontacting step.
 10. The method according to claim 9, wherein thenucleic acid ligand is single stranded.
 11. The method according toclaim 9, wherein the nucleic acid ligand is DNA.
 12. The methodaccording to claim 9, wherein the nucleic acid ligand is RNA.
 13. Themethod according to claim 9, wherein the nucleic acid ligand comprises anucleotide sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO:
 2. 14. The method according to claim 9, wherein thenucleic acid ligand comprises an RNA sequence transcribed from anucleotide sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO:
 2. 15. The method according to claim 9, wherein thesubject is a human.
 16. The method according to claim 15, wherein thetransmissible spongiform encephalopathy disease is selected from thegroup consisting of: Creutzfeldt-Jakob Disease (CJD); VariantCreutzfeldt-Jakob Disease (vCJD); Gerstmann-Straussler-ScheinkerSyndrome; Fatal Familial Insomnia; and Kuru.
 17. The method according toclaim 9, wherein the subject is an animal.
 18. The method according toclaim 17, wherein the transmissible spongiform encephalopathy disease isselected from the group consisting of: Bovine Spongiform Encephalopathy(BSE); Chronic Wasting Disease (CWD); Scrapie; Transmissible minkencephalopathy; Feline spongiform encephalopathy; and Ungulatespongiform encephalopathy. 19-36. (canceled)